Composition exhibiting a von willebrand factor (vWF) protease activity comprising a polypeptide chain with the amino acid sequence AAGGILHLELLV

ABSTRACT

The invention relates to vWF cleaving entities having a molecular weight of 180 kD, 170 kD, 160 kD, 120 kD or 110 kD and an N-terminal amino acid sequence of AAGGILHLELLV, vWF cleaving complexes and methods for their production.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/721,254 filed on Nov. 22, 2000.

FIELD OF THE INVENTION

[0002] The invention relates to a vWF protease-containing compositionwhich includes a polypeptide comprising the amino acid sequenceAAGGILHLELLV, as well as to nucleotide sequences coding for such apolypeptide. It further relates to methods for increasing the stabilityof the vWF protease.

BACKGROUND OF THE INVENTION

[0003] vWF is a glycoprotein circulating in plasma as a series ofmultimers ranging in size from about 500 to 20,000 kD. Multimeric formsof vWF are composed of 250 kD polypeptide subunits linked together bydisulfide bonds. vWF mediates the initial platelet adhesion to thesubendothelium of a damaged vessel wall, though only the largestmultimers appear to exhibit haemostatic activity. Such vWF multimershaving large molecular masses are stored in the Weibel Palade bodies ofendothelial cells, and it is believed that endothelial cells secretethese large polymeric forms of vWF. Those forms of vWF which have a lowmolecular weight (low molecular weight or LMW vWF) are believed to arisefrom proteolytic cleavage of the larger multimers.

[0004] A small portion of the vWF present in normal plasma circulates as189, 176 and 140 kD fragments resulting from proteolytic degradation ofvWF in vivo, the 140 kD fragment being derived from the N-terminalregion, and the 176 kD fragment from the C-terminal region of thesubunit. When LMW forms of vWF are isolated from normal human plasma andsubjected to SDS-PAGE (polyacrylamide gel electrophoreses) afterdisulfide reduction, an unusually high portion of vWF fragments arefound. This finding is compatible with the view that LMW forms of vWFhave been partially or predominantly derived from large multimers byproteolytic degradation.

[0005] The proteolytic degradation of vWF is a physiological process inhealthy individuals, yet in patients suffering from von Willebranddisease (vWD) type 2A it may be accelerated, and as a consequence thesepatients lack the vWF multimers with the largest molecular masses. Alack of large vWF multimers and an increased level of proteolyticfragments are also observed in acquired von Willebrand disease (vWD)associated with myeloproliferation syndrome, indicating increased invivo proteolysis in this condition as well.

[0006] In patients with thrombotic thrombocytopenic purpura (TTP), onthe other hand, unusually large vWF multimers are detected, andincreased vWF binding to platelets has been demonstrated in thesepatients (Moake et al., New Engl.J.Med., 1982, 307, pp. 1432-1435).Familial TTP is associated with a severe congenital deficiency of vWFprotease, while the presence of vWF-cleaving proteases inhibitingautoantibodies has been observed in patients with non-familial TTP.

[0007] The large multimers of vWF associated with TTP normally disappearafter a patient is transfused with normal fresh frozen plasma.Presently, plasma exchange is the most important treatment for TTP,although significant side effects have been reported with this therapy.The existence of a severe congenital deficiency of vWF protease has beenestablished in patients with familial TTP and the presence of avWF-cleaving protease inhibiting autoantibodies has been observed inpatients with non-familial TTP.

[0008] Several proteases have been shown to be able to cleave vWF,thereby impairing its binding affinity for platelets. However, in vitrothe cleavage of vWF with these proteases in each case results incleavage products different from the fragments derived from in vivocleavage.

[0009] Thus, for example, while plasmin is capable of cleaving severalpeptide bonds in vWF, plasmin-treated vWF retains a high molecularweight core region retaining about 70% of its platelet agglutinatingactivity (determined as ristocetin cofactor). A 34 kD peptide is splitfrom the N-termini of individual vWF subunits in the early stages ofplasmin treatment, and epitope mapping of such plasmin-induced fragmentsshow that these fragments originated from regions of the vWF subunitthat are different from the vWF fragments present in circulating plasma.

[0010] Porcine pancreatic elastase and various serine proteases releasedfrom human leukocytes have also been shown to degrade vWFproteolytically with a resultant loss of large multimers. Epitopemapping of the degradation products again indicates that these fragmentsalso differ from those present in normal plasma and in vWD type 2A. Inaddition, a calpain-like protease released from human platelets has beenshown to degrade large vWF multimers and to create vWF fragments similarto those observed in vivo.

SUMMARY OF THE INVENTION

[0011] We have isolated a composition exhibiting vWF protease activitythat is capable of proteolytically processing vWF in a physiologicalmanner. Said composition comprises at least one single peptide chainhaving a molecular weight between 190 kD and 100 kD as determined by SDSPAGE and comprises the sequence AAGGILHLELLV. This amino acid sequenceis located at the N-terminus of the peptide chain. The compositioncomprising the sequence AAGGHILHLELLV can also be used for isolation,detection or purification of proteins, i.e. von Willebrand Factor.

[0012] Furthermore, an isolated polypeptide having a molecular weightbetween 190 kD and 100 kD according to SDS-PAGE and comprising thesequence AAGGILHLELLV is also provided. This sequence is preferablydirectly followed by the sequence AVG, which is preferably followed bythe sequence PDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI.

[0013] Another aspect of the present invention is a method of purifyingvon Willebrand factor comprising contacting a solution containing vonWillebrand factor with a substrate comprising the amino acid sequenceAAGGILHLELLV under conditions sufficient to bind von Willebrand factorto the substrate.

[0014] The present invention further comprises a method of treatingthrombotic diseases using a polypeptide of the present invention, asshown in FIG. 2.

[0015] In addition, the present invention includes a method ofprocessing recombinantly produced vWF through the use of the vWFprotease of the present invention, in order to produce a vWF product.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows the schematic purification scheme of the vWF proteasecontaining composition of the present invention.

[0017]FIG. 2 shows the partial nucleotide and amino acid sequence of thevWF protease of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0018] A composition is provided containing a vWF protease consisting ofa polypeptide chain with an apparent molecular weight in SDS-PAGE ofaround 180 kD, around 170 kD, around 160 kD, around 120 kD, around 110kD or mixtures of these chains, said chain comprising an amino acidsequence AAGGILHLELLV. Alternatively, this amino acid sequence can bedirectly followed by the amino acid sequence AVG. Furthermore, thissequence is followed by the sequencePDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI.

[0019] The SDS-PAGE was performed under reducing conditions. As is wellknown in the art, molecular weight determination using SDS PAGE resultsin the detection of apparent molecular masses, which may be differentfrom the molecular masses of the native, non-denatured protein.

[0020] Based on a computer search of sequence homologies, the vWFprotease according to the invention shows high homology to the group ofdisintegrin and metalloproteinases (ADAM). Members of this group shareseveral distinct protein modules, including a protease domain, adisintegrin domain, a cysteine-rich region and an EGF repeat (Tang B Land Hong W., FEBS, 1999, 445, pp. 223-225). The nucleotide and aminoacid sequence as shown in FIG. 2 comprises at least 4 exons of theprotease portion of the vWF protease of the present invention.

[0021] Analysis of non-denatured material by mass spectrometry showedvery broad peaks of high molecular weight. No prominent bands wereidentified. This finding is in agreement with appearance, in gelfiltration experiments, of 80 kD bands close to the void volume ofSephacryl® S-300, suggesting that the proteins in this preparation tendto polymerize under physiologic conditions (a property of clustering.

[0022] The AAGGILHLELLV sequence is located at the N-terminal region ofthe protein. Shortening of the peptide chain occurs at the C-terminus orvia endoproteolytical cleavages. The composition according to thepresent invention contains a vWF cleaving protease that is expressed asa single chain protein.

[0023] Preferably, the composition according to the present inventionfurther comprises Ca²⁺, Sr²⁺ and/or Ba²⁺ ions. The preparation maycomprise calcium ions in a concentration of about 1 to 10⁶ ions perpolypeptide molecule with vWF protease activity. The preparationaccording to the present invention contains vWF protease activity in anessentially purified form. Preferably, the purified protease preparationfrom plasma contains between 0.001% and 1%, preferably 0.002% of theinitial amount of plasma protein, and between 1% and 5%, preferably 2.3%of the initial enzyme activity, which has been partially inactivatedduring the purification procedure. Preferably, the purity is as high asthe relative proportions of polypeptide chains with the vWF proteaseactivity present in an amount of above 50%, especially above 80%, mostpreferred about 90%, of total protein compared to the vWF proteaseactivity in plasma. Preferably, the preparation according to the presentinvention is essentially free of vWF or vWF fragments, i.e. having a vWFcontent of below 5%, preferably below the detection limit of an assayused to detect vWF.

[0024] The peptides containing the amino acid sequence MGGILHLELLV orAAGGILHLELLVAVG or AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI or RRAAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI can also be used as tool for detecting proteins bindingto the vWF protease or target sites for ligand development fordetecting, isolating and purifying proteins that bind to the vWFprotease. Preferably the protein to be detected or purified is vWF.

[0025] These ligands can for example be peptides or peptidomimeticscapable to bind proteins binding to the vWF protease or binding domainsincorporated into antibodies or antibody derivatives (for example singlechain antibodies, miniantibodies, bispecific antibodies, diabodiesetc.).

[0026] Furthermore, the peptide having the amino acid sequenceMGGILHLELLV or MGGILHLELLVAVG or AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI or RRAAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI can also be used for the development of anti-vWFprotease antibodies using techniques as known from the art. Thedevelopment of these antibodies or antibody derivatives orpeptidomimetics can be accomplished according methods known to the priorart (Greer J. et al., J.Med.Chem., 1994, Vol. 37, pp. 1035-1054; HarlowE. and Lane D., in “Antibodies. A Laboratory manual”, Cold Spring HarborLaboratory, 1988, Esser C. and Radbruchj A., Annu. Rev. Immunol., 1990,vol. 8, pp. 717-735; Kemp D. S., 1990, Trends Biotechnol., pp. 249-255).

[0027] The present invention relates also to single polypeptide chainshaving an apparent molecular weight in reduced SDS-PAGE of between 190kD and 100 kD, preferably about 180 kD, more preferably about 170 kD, ina particularly preferred embodiment about 160 kD, preferably 120 kD andmost preferably about 110 kD, comprising an N-terminal amino acidsequence AAGGILHLELLV. This sequence is preferably directly followed bythe sequence AVG, which is then preferably followed by the sequencePDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI. In contrastto the protease entities described by Furlan et al. (1996) and Tsai(Blood 87(10) (1996), pp.4235-4244) the vWF multimerase entitiesaccording to the present invention are much smaller than the entitiesdescribed in these documents (around 300 kD (Furlan et al.), and 200 kD(Tsai), respectively).

[0028] The proteolytic entities provided with the present inventioncomprise a physiological vWF cleaving activity which is defined by (1)the cleaving vWF at the peptide bond 842Tyr-843Met, (2) having a directproteolytic activity which converts vWF having a singlet structure tovWF having a satellite structure, and (3) retaining activity in thepresence of a serine protease inhibitor such as diisopropylfluorophosphate (DFP) and in the presence of a calpain proteaseinhibitor such as carbobenzyloxy (Z) peptidyl diazomethylketoneinhibitor (Z-Leu-Leu-Tyr-CHN₂). The proteolytic entities provided withthe present invention may also act indirectly via another effectorprotein, for example a protease.

[0029] This single polypeptide chain forms an active vWF cleavingcomplex together with a metal ion selected from the group consistingCa⁺⁺, Sr⁺⁺ and Ba⁺⁺. The preferred metal ion is Ca⁺⁺. This activecomplex is able to cleave vWF in a physiological manner as describedabove.

[0030] A further aspect of the present invention relates to an isolatedvWF cleavage complex comprising vWF, a metal ion selected from the groupconsisting of Ca⁺⁺, Sr⁺⁺ and Ba⁺⁺ and one or more single polypeptidechains having vWF protease activity according to the present invention.

[0031] The vWF proteolytic activity (also termed “vWF proteaseactivity”) of the peptide chains according to the present invention maybe determined by any method described in the art, such as the methodaccording to Furlan et al. (1996), which is preferred for the presentinvention. Further preferred test systems are described in InternationalApplication No. WO 00/50904. The test system described in WO 00/50904 isalso suitable for the present vWF protease entities. Alternatively, acollagen binding assay (as described in EP 816 852) can also be used asa test system. Preferably, this test is used for screening anddiagnostic purposes.

[0032] A further object of the invention is achieved by providing acomposition containing the vWF protease according to the inventiontogether with clusterin or an analog or derivative thereof. Withrelation to the activity of the protein, the term “derivative” or“analog” of clusterin, refers to proteins that show the same proteolyticcharacteristics as the native clusterin protein.

[0033] Clusterin is a heterodimeric glycoprotein consisting of twonon-identical subunits, with a molecular mass of approximately 80 kDa(Rosenberg and Silkensen, J., Int.J.Biochem. Cell Biol., 1995, vol. 27,pp. 633-645; Tschopp J. and French, L.E., Clinical and Exp. Immunol.,1994, 97, pp. 11-14). It is produced in a wide array of tissues andfound in most biologic fluids. The physiologic functions described inthe prior art include complement regulation, lipid transport, spermmaturation, initiation of apoptosis, endocrine secretion, membraneprotection and promotion of cell interactions.

[0034] It has been found that the unusually high stability of the vWFprotease of the present invention in circulating plasma is associatedwith the presence of clusterin. We have found that the half-life ofvWF-cleaving protease activity in vivo is between 1 and 4 days, whileother proteases in plasma have half-lives in the range of seconds tohours. The ratio of clusterin to vWF protease in a composition accordingto the present invention is preferably in a range of 10M:1M to 1M:10M,and more preferably the ratio of clusterin and vWF is in the equimolarrange. In human plasma, the concentration of vWF-cleaving protease is2-10 mg/liter whereas that of clusterin is 50-400 mg/liter plasma (themolar ratio of vWF-cleaving protease to clusterin in human plasma isabout 1:20-1:100).

[0035] Isolation of the composition from either human plasma or othersources, e.g. supernatants of cell cultures expressing the polypeptideaccording to the present invention, milk or other body fluids oftransgenic animals expressing the polypeptide according to the presentinvention can be performed by chromatographic means. Preferably, thepurification is performed by a combination of chromatographic stepsincluding immunoaffinity chromatography, gel filtration, and ionexchange chromatography. For example, the first purification step can beimmunoaffinity chromatography, the second step can be gel filtration,followed by one or more additional immunoaffinity chromatography steps.A further purification can be performed using ion exchangechromatography, preferably anion exchange chromatography and at leastone affinity chromatography. Further purification steps can be performedusing ion exchange chromatography, gel filtration and further affinitychromatography steps.

[0036] As an alternative embodiment, the nucleotide sequence as shown inFIG. 2 can be used for constructing expression systems providingappropriate elements for the expression of the DNA which can then beused for the expression of a polypeptide having vWF protease activityaccording to the present invention.

[0037] The expression vector may comprise, for example, in the directionof transcription, a transcriptional regulatory region and atranslational initiation region functional in a host cell, a DNAsequence encoding for the polynucleotide expressing a VWF proteaseactivity according to the present invention and translational andtranscriptional termination regions functional in said host cell,wherein expression of said nucleic sequence is regulated by saidinitiation and termination regions. The expression vector may alsocontain elements for the replication of said nucleotide. Examples of DNAexpression vectors are pBPV, pSVL, pRc/CMV, pRc/RSV, myogenic vectorsystems (WO 93/09236) or vectors derived from viral systems, for examplefrom vaccinia virus, adenoviruses, adeno-associated virus, herpesviruses, retroviruses or baculo viruses.

[0038] The expression vector containing the nucleic acid which encodesthe polypeptide having vWF protease activity according to the presentinvention can be used to transform host cells which then produce saidpolypeptide. The transformed host cells can be grown in a cell culturesystem to produce said polypeptide in vitro. The host cells preferablyexcrete the polypeptide having vWF protease activity into the cellculture medium from which it can be prepared.

[0039] The host cells may be cells derived from the body of a mammal,for example fibroblasts, keratinocytes, hematopoietic cells, hepatocytesor myoblasts, which are transformed in vitro with an expression vectorsystem carrying a nucleic acid according to the present invention andre-implanted into the mammal. The polypeptide according to the presentinvention encoded by said nucleic acid will be synthesized by thesecells in vivo and they will exhibit a desired biological activity in themammal.

[0040] The nucleic acid encoding the polypeptide according to thepresent invention may also be used to generate transgenic animals, whichexpress said polypeptide proteins in vivo. In one embodiment of thisspecific application, the transgenic animals may express the polypeptidehaving vWF protease activity in endogenous glands, for example inmammary glands from which the said proteins are secreted. In the case ofthe mammary glands, said proteins having vWF protease activity aresecreted into the milk of the animals from which said proteins can beprepared. The animals may be mice, cattle, pigs, goats, sheep, rabbitsor any other economically useful animal.

[0041] The vWF protease composition of the present invention can beused, for example, to process recombinantly produced vWF. RecombinantvWF (r-vWF) can be produced in CHO cells, e.g. according to FEBS Letter375, 259-262 (1995). The r-vWF recovered in this manner is available asa mature vWF and has a singlet structure, i.e. it differs fromplasma-derived vWF, which always has a characteristic satellitestructure when examined on 2% SDS agarose gels. In International PatentApplication No. WO 96/10584 describes that r-vWF is comprised ofmultimers with high structural integrity which is retained even afterpurification and treatment for the inactivation of viruses. The intactstructure of the r-vWF is defined by a result of electrophoreticanalysis consisting of multimer bands with an absence of satellitebands. To prepare an r-vWF preparation having a structure more closelycorresponding to that of plasma-derived vWF from r-vWF with singletstructure, r-vWF is treated with the vWF protease composition of thepresent invention.

[0042] According to a further aspect of the present invention, the aminoacid sequence and nucleotide sequence as shown in FIG. 2 can also beused for the production of a preparation for the prophylaxis and therapyof diseases that show supranormal vWF content or an increased level ofhigh-molecular weight vWF in patients. This can result in thromboses andthromboembolic diseases. For example, thrombotic throbocytic purpura(TTP), Henoch-Schönlein purpura, preeclampsia, neonatal thrombocytopeniaor haemolytic-uremic syndrome. By administering an effective dose of apolypeptide having a vWF protease activity and having an amino acidsequence as shown in FIG. 2, this can lead to reduction of the contentof high molecular weight vWF multimers in the patients, resulting ineffective therapy of these diseases. The invention is described in thefollowing examples, without being limited thereto.

EXAMPLES Example 1 Method for Isolating vWF-Cleaving ProteolyticPeptides

[0043] 1.1. Preparation of an IgG-eTTP-coupled affinity gel. TheIgG-eTTP was isolated by aid of a 20 ml protein A-Sepharose® (diameter1.6 cm) in TBS, pH 7.4. Pheresis plasma of a patient suffering fromacquired TTP (“erworbenes” TTP; eTTP), which previously had been assayedfor its inhibitor content relative to the vWF-cleaving protease wasapplied to the column in a volume of 50 ml. After subsequent rinsingwith TBS, pH 7.4, the bound IgGs were step-wise eluted with citrate, 0.1M, pH 4.0, and glycine, 0.1 M, pH 2.7. The fractions immediately werebrought to a physiological pH by adding Tris, 1.5 M, pH 8.8, anddialysed against TBS, pH 7.4. The Affi-Gel® Hz was coupled according tothe producer's instructions with the IgG-eTTP which had been washed outof the protein A-Sepharose® with a pH of 4.0. The column materialprepared in this manner first was washed as prescribed, subsequently itwas washed 3 times alternatingly with 50 ml of buffer B and 200 ml ofbuffer A (chapter 1.7). Prior to use, intensive rinsing with buffer Awas carried out in each instance.

[0044] 1.2 First Step

[0045] As the starting material, 100 ml of pooled CPD plasma which hadcome from at least three donors and had been stored at −20° C., was usedafter centrifuging at 2,500 rpm (1,100 g) for 5 min. At a relatively lowflow rate (FR: 30 ml/h), the plasma was loaded on a 200 mlchromatographic column with IgG-eTTP Affi-Gel® Hz (hydrazide, diameter2.6 cm) which had been equilibrated in buffer A. After washing with atleast 400 ml of buffer A over night at the same flow rate, a 200 mldesalting gel filtration column (Bio-Gel® P-6DG, diameter 2.6 cm) and a10 ml protein G-Sepharose® (diameter 1.6 cm), which previously also hadbeen rinsed with buffer A, was connected thereto. After the flow ratehad been increased to 100 ml/h, the proteins bound to Affi-Gel Hz wereeluted directly with 50 ml of buffer B onto the Bio-Gel® P-6DG so as toremove from the proteins the NaSCN that had been in buffer B. Theproteins which had been eluted from the desalting column prior to theNaSCN were led through the protein G Sepharoseo without interruption,where they were freed from the IgGs. Here, the flow rate was lowered to50 ml/h so as to extend the dwell time of the proteins in the 10 mlcolumn. For regeneration, the protein G-Sepharose® was shortly washedwith buffer C, and the eluted IgG fraction was stored for analysis.

[0046] The first step was carried out 8 times before the collectedfractions which had been frozen at −20° C. were pooled and furtherprocessed.

[0047] 1.3 Second Step

[0048] The pooled fractions from 8 chromatographies of the first stepwere diluted 1:1 with H₂O so as to obtain an ionic strength at which thedesired proteins would bind to the anion exchange column (High QSupport®). The sample whose volume was from 1,500 to 1,800 ml, dependingon the charge used, was checked for its pH and its ionic strength andapplied over night at a FR of 90 ml/h through a 50 ml column withTherasorb® (diameter 1.6 cm) onto a 5 ml High Q Support® (diameter 1.6cm). Both, Therasorb and High Q Support® had previously beenequilibrated in buffer D. After washing with approximately 150 ml ofbuffer D, the Therasorb was disconnected, and the 25 ml Lentil LectinSepharose® (diameter 1.6 cm) which had been equilibrated in buffer E wasconnected to follow the High Q Support®. At a FR of 60 ml/h, theproteins bound to High Q Support were immediately eluted with buffer Edirectly to the Lentil Lectin-Sepharose®. The proteins which bound tothe Lentil Lectin-Sepharose® could be eluted in two steps with buffers Gand H and could be collected. The proteins which had remained bound toTherasorb and High Q Support® were washed out with buffer C or buffer F,respectively, and discarded after an analysis.

[0049] For regeneration, before being used, the Lentil Lectin-Sepharose®in each case was rinsed according to the producer's instruction 3 timesalternatingly with 20 ml each of buffers I and J, the High Q Support wasrinsed successively with 10 ml each of NaOH 1N and NaCl 1M.

[0050] 1.4 Third Step

[0051] The pooled fractions which had been eluted from the LentilLectin-Sepharose® with buffer H were dialysed three times for a total of4 h, each against 1 l of buffer D, and again applied to the High QSupport at a flow rate of 60 ml/h. Connected thereinfront was a 5 mlheparin-Sepharose (diameter 1.4 cm), which likewise had beenequilibrated in buffer D. After the application of the sample, it wasrinsed with approximately 50 ml of buffer D, the heparin-Sepharose wasdisconnected, and a 500 ml Sephacryl® S-300 HR (diameter 2.6 cm), whichhad been equilibrated in buffer L, was connected thereto. The proteinsbound to High Q Support were directly eluted to the gel filtrationcolumn with 10 ml of buffer K. The exclusion chromatography was effectedat a flow rate of 42 ml/h, and the fractions were collected at 7 mleach. The proteins which were more strongly bound to High Q Support®were again eluted with buffer F, those which remained adhered to theheparin-Sepharose, with buffer K.

[0052] 1.5 Fourth Step

[0053] The pool of the active fractions from the third step was appliedwithout treatment at a FR of 10 ml/h to a 1 ml anti-α₂-macroglobulincolumn (flow rate 0.7 cm) which had been equilibrated in buffer L. Theanti-α₂-macroglobulin column was prepared by immobilization according tothe instructions, of rabbit-anti-α₂-macroglobulin antibodies at aconcentration of 4.9 mg/ml on CNBr-activated Sepharose. The proteinsbound thereon were eluted with NaSCN 3M in buffer L and with buffer Cand stored for analysis. Materials buffer A Tris 10 mM pH 7.4 NaCl 0.15M Na₃-citrate 1 mM Na acid 0.02% buffer B NaSCN 3.0 M pH 7.4 in buffer Abuffer C glycine 0.1 M pH 2.7 Na acid 0.02% buffer D Tris 10 mM pH 7.4NaCl 75 mM buffer E Tris 20 mM pH 7.4 NaCl 0.5 M MnCl₂ 1 mM buffer FTris 10 mM pH 7.4 NaCl 1.0 M buffer G Tris 20 mM pH 7.4 NaCl 0.5 MMethyl-α-D-mannopyranoside 30 mM buffer H Tris 20 mM pH 7.4 NaCl 0.5 MMethyl-α-D-mannopyranoside 0.3 M buffer I Tris 20 mM pH 8.5 NaCl 0.5 Mbuffer J Na acetate 20 mM pH 5.5 NaCl 0.5 M buffer K Tris 10 mM pH 7.4NaCl 0.5 M buffer L (TBS) Tris 10 mM pH 7.4 NaCl 0.15 M

[0054] Chromatographic materials affi-gel hydrazide gel ®: forimmobilizing specific Bio-Rad, Hercules, CA, USA IgG'santi-α₂-macroglobulin column: isolation of applicant's own production(see 1.5): α₂-macroglobulin rabbit-anti-human-α₂-macroglobulin antibodyon CNBr activated Sepharose 4B; 4.9 mg/ml Bio-Gel ® P6-DG, medium: gelfiltration Bio-Rad with exclusion limit ≧ 6 kDa CNBr activated Sepharose4B ®: for Amersham Pharmacia Biotech, Uppsala, immobilizing proteins Sheparin Sepharose, HITrap ® 5 ml: Amersham Parmacia affinitychromatography: binds various proteins High Q Support ®, Macro-Prep:strong Bio-Rad anion exchanger IgG-eTTP Affi-gel Hz: for bindingvWF-protease applicant's own production (see 1.1.): IgG- eTTP onAffi-gel Hz hydrazide Lentil Lectin-Sepharose 4B: affinit AmershamPharmacia chromatography: binds to sugar residues o proteins protein ASepharose ® CL-4B. binds IgG of Amersham Pharmacia type 1, 2 and 4protein G Sepharose ® 4FF: isolation of IgGs Amersham Pharmacia of alltypes Sephacryl ® S-300 HR: gel filtration for MWs Amersham Pharmacia10,000 to 1,500,000 Therasorb: coupled with sheep-anti-human-IgSerag-Wiessner, Naila, D antibodies: isolation of human immunoglobulins

[0055] 1.6 Fifth Step

[0056] Alternatively, or in addition to step four, an anti-clusterincolumn chromatography as a further step can be applied. The samples wereprepared identically to the anti-α2-macrogluobulin-column usinganti-clusterin antibodies.

[0057] 2. SDS-Page Reduced/Non-Reduced

[0058] SDS-Polyacrylamide Gel-Electrophoresis (SDS-PAGE)

[0059] SDS-PAGE was done according to Lämmli. The separating gels wereprepared having a size of 13.5 cm height, 15 cm width and 3 mmthickness, having the composition as follows: Concentration ofSDS-Polyacrylamide gel for a gradient gel of 4% to 12%: Acrylamide 4%12% N,N′-Methylenebisacrylamide 0.107% 0.32% Tris(Tris(hydroxymethyl)aminomethane) 0.4 M 0.4 M APS(Ammoniumperoxydisulfate) 0.03% 0.03% SDS (Sodium Dodecyl Sulfate) 0.1%0.1% Temed 0.067% 0.067% (N,N,N′,N′-Tetramethylethylenediamine) pH 8.7pH 8.7

[0060] 30 ml of each of the 4% and 12% solutions were poured between twoglass plates.

[0061] After polymerization, the stacking gel was prepared, having aheight of 3 cm and having 16 slots. The volume of each slot was 150 μl.Stacking gel: Acrylamide 3% N,N′-Methylenebisacrylamide 0.08% Tris 0.1 MAPS 0.03% SDS 0.1% Temed 0.2% pH 6.8

[0062] 100 μl probe were mixed with 50 μl buffer for the SDS-PAGE andincubated 20 minutes at 60° C. Probes that had to be reduced were mixedwith DTT (1,4-Dithio-DL-threitol), 65 mM before the solution was heatedup. Buffer for the SDS-PAGE: Tris 0.15 M SDS 4% Glycerin-87% 30% Brominephenol blue few amounts pH 6.8

[0063] After heating, the probes were centrifuged, loaded onto the slotsof the SDS PAG and covered with electrophoresis buffer. Electrophoresiswas run over night in a vertical chamber system, using 60 V voltage.Electrophoresis buffer: Tris 50 mM Glycine 0.38 M EDTA (Titriplex III:ethylen- 2 mM dinitrilo tetra acetic acid- disodium salt -dihydrate) SDS0.1% standard-pH 8.3

[0064] Silver Staining:

[0065] The gels (13.5×15×0.3 cm) were put into a tray containing amixture of methanol (25%), acetic acid (7.5%), water (65.5%) andglycerin-87% (2%) (fixing solution) for at least 3 hours. Subsequently,the gels were rinsed 4-times with 200 ml water for 1 hour. Then the gelwas put into a sodium thio sulfate solution (0.02%), washed twice withwater (1 min) and incubated 2-times 20 minutes with silver nitrate(0.1%) in water under constant shaking.

[0066] After rinsing with water for two times, the gel was shaken in adeveloping solution, consisting of sodium carbonate (2.5%) andformaldehyde-37% (0.04%) in water. The reaction was stopped with thefixing solution, as soon as the proteins were visible as gray to brownbands.

Example 2 Affinity Purification of von Willebrand Factor (vWF)

[0067] The peptide with the sequence AAGGILHLELLV was synthesized on asolid-phase support following the method of Barany, G and Merrifield, R.B. (1980) Solid-phase Peptide Synthesis, in The Peptides vol. 2 (Gross,E. and Meienhofer, J., eds) Academic, New York. After cleavage andde-protection of the peptide, the peptide was purified by ion-exchangechromatography. The peptide was characterized by reverse phase HPLC on aC8 silica column with gradient elution in trifluoro acetic acid withacetonitrile. The peptide showed no major byproducts.

[0068] The peptide was solubilized in a concentration of 5 mg/mL in 0.1molar phosphate buffer pH 7.5 and incubated with a pre-activated gelsuitable for affinity chromatography (Actigel, ALD-Superflow,Sterogene). Prior to coupling of the peptide to the gel thepre-activated matrix was excessively washed with the same phosphatebuffer. One volume of the pre-washed gel was then mixed with one volumeof the peptide solution to be immobilized and subsequently 0.1 volumeportions of a solution of 0.1 molar cyanoborohydride (NaCNBH₃) in 0.1molar phosphate buffer pH 7.5. The gel was suspended in this solutionand shaked for 15 hours at room temperature. Subsequently the gel waswashed on a sinter funnel with a 10-fold volume of the phosphate buffercontaining 150 mmolar NaCl and with 5 volumes of the phosphate buffercontaining 2 molar NaCl. Then the gel was equilibrated with an access of0.1 molar phosphate buffer pH 7.0.

[0069] The gel was then transferred into a chromatographic column havinga dimension of diameter to gel bed height of 1:4. By determining thepeptide concentration the solution of the incubation supernatant afterseparation from the gel and the washing solutions the amount of peptidecoupled to the affinity matrix was calculated. The coupling rate was85%.

[0070] The gel was subsequently used to purify vWF from a Factor VII(FVIII)/vWF complex. A FVIII/vWF complex concentrate was producedaccording to EP 0 270 516 containing vWF in a concentration of 260 UvWF:Ag/ml and a specific activity of 13.5 U vWF:Ag/mg Protein. Theconcentrate was diluted with 20 mM phosphate buffer pH 7.0 to a finalvWF concentration of 6 U vWF:Ag/mL. A volume of 20 ml of this solutionwas subjected to the affinity column with immobilized peptide describedabove. After washing the column with 10 ml of the phosphate buffer thevWF specifically bound to the peptide ligand was eluted by a lineargradient from 0-2 mol/l NaCl in phosphate buffer at a flow rate of 1ml/minute. Fractions of 1 ml were collected and their optical densitywas determined at 280 nm. All fractions were measured for their contentof vWF antigen determined by a specific ELISA method (Asserachrom vWF,Boehringer, Mannheim). Measurement showed a specific peak of vWF elutingfrom the peptide at a NaCl concentration of 100 mmol/l, while most ofthe protein measured by UV-absorption eluted prior to the vWF fractionwith the washing buffer. The vWF containing fractions were pooled andmeasured for vWF activity. The vWF in this pool had a specific activityof 95 U vWF/mg protein and was essentially free from other proteins.

Example 3 Anti-Peptide Antibodies

[0071] The peptide with the sequence MGGILHLELLV was synthesized andpurified as described in example 2. The peptide was then used toimmunize 3 months old BALB/c mice with the following protocol: A primarysubcutaneous injection of 100 μg peptide antigen emulsified in Freund'scomplete adjuvant in 100 μl followed by intra-peritoneal boosts of 100μg peptide antigen in phosphate buffered saline at monthly intervals.

[0072] The anti-peptide titer was tested by routine ELISA method usingpurified peptide as screening antigen. After the final boost the spleenswere taken from the mice for cell fusion. Cell fusion was carried outaccording to a standard protocol originally described by Kohler G. andMilstein C. 1975, Nature 256:495. Anti-peptide antibodies producinghybridoma cell lines were screened by standard techniques with thepurified peptide as screening antigen essentially based on aconventional ELISA methodology. After cloning a cell line could beisolated with a high expression level of an antibody specific for thescreening peptide with the sequence AAGGILHLELLV. This cell line wascultured on serum-free culture medium and grown to high density. Thesupernatant of the cell culture was harvested by centrifugation toremove cells and the monoclonal antibody containing supernatant wasconcentrated by ultra-diafiltration and conditioned for further use.

[0073] The monoclonal antibody obtained had a high selectivity for thevWF cleaving protease as described by Furlan et al. 1996, Blood87:4223-4234. This monoclonal antibody was immobilized to a polystyreneELISA plate in a carbonate/bi-carbonate buffer, 0.05 molar, pH 9.6, at aconcentration of 5 μg immunoglobuline/ml overnight (16 hours) at 4° C.,with each 100 μl of coating solution per well. The coating solution wasremoved from the wells and replaced by a solution of bovine serumalbumin (BSA) at a concentration of 100 μg/ml at a volume of 100 μL perwell, for 2 hours. The BSA solution was removed and the wells werewashed with phosphate buffered saline. The pre-coated plates were thenincubated with either samples of platelet poor plasma from healthy humanplasma donors or platelet poor plasma from patients with an uncleardiagnosis of either thrombotic thrombocytopenic purpura (TTP) orhemolytic uremic syndrome (HUS). After incubation of the plasma sampleswith the antibody coated ELISA plates as in a routine sandwich ELISAsystem, after 3 hours the plasma was removed from the wells. Wells werewashed with phosphate buffered saline and incubated with the monoclonalantibody directed against the peptide with the sequence AAGGILHLELLV,conjugated with horse radish peroxidase following the method of Wilson,M. B. and Nakane, P. K. (1978) In Immunofluorescence and RelatedStaining Techniques. Knapp, W. Holubar, K. and Wick, G (eds),Elsevier/North Holland, Amsterdam, p. 215, and detected by the OPTreagent as described by Cathy D. and Raykundalia Ch. (1989) ELISA andrelated enzyme immunoassays, in Antibodies II a practical approach.Cathy D (ed), IRL Press Eynsham Oxford England, p. 97.

[0074] Based on the level of the samples from the healthy human plasmadonors a normal range was established. Plasmas from patients with HUShad a vWF protease activity equivalent to healthy humans while patientswith TTP had a decreased protease activity as confirmed by an assaybased on a different assay principle as described in WO00/509904.

Example 4 Amino Acid Sequencing and Amino Acid Analysis of the vWFProtease

[0075] The final protein preparation from the third step of isolationwas electrophoresed on a 1.5 mm-thick SDS-polyacrylamide gel accordingto Laemmli (Laemmli UK., Nature, 1970, 227, pp. 680-685) A gradient of 4to 12% polyacrylamide was used for fractionation of high molecularweight proteins, and a gradient of 8 to 12% polyacrylamide for lowmolecular weight proteins. After electrophoresis under non-reducing orreducing conditions (final concentration 65 mmol/l dithiotreitol), theproteins were blotted onto PVDF-membranes and stained for 2 min with0.25% Coomassie Blue in 45% methanol, 9% acetic acid and 46% H₂O. Afterrinsing with a mixture of 50% methanol, 10% acetic acid and 40% H₂0, thevisible protein bands were cut out and analyzed on a Procise-cLCSequencer (Foster City, CA) at the Chemical Institute of the Universityof Bern.

[0076] The N-terminal amino acid sequence of polypeptide bands separatedby SDS-PAGE of purified vWF-cleaving protease is shown in table 1:Molecular weight Amino Acid sequence 350 kDa unreducedSer-Val-Ser-Gly-Lys-Pro-Gln-Tyr-Met-Val* 150 kDa unred.Ala-Ala-Gly-Gly-Ile 140 kDa unred. Ala-Ala-Gly-Gly-Ile 130 kDa unred.Ala-Ala-Gly-Gly-Ile 110 kDa unred. Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu70 kDa unred. Asp/Ser-Gln/Leu-Thr/Met-Val/Pro- Ser/Phe** 180 kDa reducedAla-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu 170 kDa reduced Ala-Ala-Gly-Gly-Ile160 kDa reduced Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val-Gly 120 kDa reducedAla-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu- Leu-Leu-Val-Ala-Val-Gly 40 kDareduced Asp-Gln-Thr-Val-Ser**

[0077] Analysis of the composition of the amino acids was performed fromthe same sample as used for amino acid sequencing. The protein bandswere hydrolyzed in the gas phase over 6 N HCl for 22 hours at 110° C.and the amino acids were determined by high-performance liquidchromatography.

[0078] Four unreduced polypeptide bands from SDS-PAGE of purified vWF-cpwith M_(r) 150, 140, 130, and 110 kDa were analyzed. The results areshown in Tab. 2: No. residues/100 residues Amino acid 150 kDa 140 kDa130 kDa 110 kDa Asx 6.7 7.0 7.4 8.3 Glx 12.2 12.0 12.8 11.8 Ser 8.4 8.98.8 9.2 Gly 11.8 12.1 12.1 12.9 His 2.5 2.3 2.5 2.4 Arg 8.3 7.6 8.1 7.2Thr 5.6 5.5 5.6 5.7 Ala 10.1 9.5 9.6 8.2 Pro 8.9 8.5 8.3 8.1 Tyr 2.1 2.72.3 2.4 Val 7.0 6.9 6.7 6.5 Ile 2.7 2.9 2.6 3.0 Leu 10.0 9.9 9.1 9.7 Phe2.6 2.8 2.6 3.2 Lys 1.0 1.3 1.3 1.4

Example 5 Search on Chromosome 9 Clone RP11-224N20 for Potential Exonsof the vWF-Protease

[0079] The coding region of the N-terminus of the activated vWF protease(aminoacids A-A-G-G-I-L-H-L-E-L-L-V-A-V-G) was found on Chromosome 9clone RP11-224N20 bases 156653 to 156697. Thus the nucleotide sequencefrom base 150001 to 185911 was screened for potential exons. Consecutiveoverlapping genome-segments with various lengths (1500 bases-5000 bases)were analysed using search engines that were queried via theinternet-explorer. The genomic sequence segments, its translations andthe results of the search were managed using the ‘Vectors NTI Suite1 v.5.2’ computer-program (Informax Inc., USA).

[0080] The first four exons of the search were translated into thecorresponding amino acid sequence and these sequences were searched forhomologies. This search revealed that each sequence displays highhomology with the amino acid sequence of the family of human disintegrinand metalloproteinases with thrombospondin motifs (ADAM-TS).

[0081] By alignments perfomed with the ‘Vectors NTI Suite1 v.5.2-AlignX’ program (Informax Inc., USA), further potential exons wereidentified that encode for amino acid sequences that are homologous tosegments of the human ADAM-TS sequences. Exons potentially encoding forthe entire proteinase domain, for parts of the disintegrin like domain,the thrombospondin motif, the cystein-rich domain, as well as for threepotential thrombospondin submotifs of a new ADAM-TS proteinase were thusidentified. The succession of the exons on the genomic sequence wasfound to be consistant with the succession of the corresponding aminoacid segments on the ADAM-TS consensus sequence.

Example 6 Cloning of the Gene Expressing the vWF-Protease Gene

[0082] Human salivary gland poly A+ RNA was purchased from Clontech.First strand cDNA was obtained using Expand reverse transcriptase(Roche) and oligo d(T) primer according to the manufacturer'sinstructions. PCR was performed using 5′ CGGCGGGATCCTACACCTGG 3′ and 5′AATGGTGACTCCCAGGTCGA 3′ as primers with 10 ng of salivary gland cDNA astemplate and 10 U of Hot Star Taq polymerase (Quiagen). The thermalcycling parameters were an initial incubation at 94° C. for 15 minutesfollowed by 45 cycles of 94° C. (50 sec), 50° C. (50 sec), 72° C. (2min). PCR products were directly sequenced in both directions using theBigDye Terminator Cycle Sequencing Ready Reaction Kit (PerkinElmer LifeScience).

[0083] The obtained DNA sequence was used to scan the genomic data baseusing BLAST (basic local alignment search tool) programs and matched tothe chromosome 9 clone RP11-224N20. DNA sequence was translated to aminoacids sequence using ExPASy proteomic tools. The DNA and translatedamino acid sequences corresponding to 4 putative exons of chromosome9q34 are shown in FIG. 2.

1. A composition exhibiting vWF protease activity comprising at leastone single peptide chain having a molecular weight between 190 kD and100 kD as determined by SDS-PAGE and comprising the amino acid sequenceAAGGILHLELLV.
 2. A composition according to claim 1 wherein saidsequence is located at the N-terminus of the peptide chain.
 3. Acomposition according to claim 1 wherein said peptide chain has amolecular weight of about 180 kD.
 4. A composition according to claim 1wherein said peptide chain has a molecular weight of about 170 kD.
 5. Acomposition according to claim 1 wherein said peptide chain has amolecular weight of about 160 kD.
 6. A composition according to claim 1wherein said peptide chain has a molecular weight of about 120 kD.
 7. Acomposition according to claim 1 wherein said peptide chain has amolecular weight of about 110 kD.
 8. A composition according to claim 1wherein said composition cleaves vWF at the peptide bond 842Tyr-843Met.9. A composition according to claim 1 wherein said composition retainsactivity in the presence of a serine protease inhibitor and a calpainprotease inhibitor.
 10. A composition according to claim 9, wherein saidserine protease inhibitor is diisopropyl fluorophosphate.
 11. Acomposition according to claim 9, wherein said calpain proteaseinhibitor is Z-Leu-Leu-Tyr-CHN₂.
 12. A composition according to claim 1wherein said peptide chain further comprises the amino acid sequenceAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI following thesequence AAGGILHLELLV.
 13. A composition according to claim 1, furthercomprising Ca²⁺, Sr²⁺, or Ba²⁺ ions.
 14. A composition according toclaim 1 comprising Ca²⁺ ions in a concentration of about 1 to 10⁶ perselected polypeptide molecule.
 15. A composition according to claim 1,wherein said composition is essentially free of vWF or vWF fragments.16. A composition according to claim 1, further comprising clusterin oran analog or derivative thereof.
 17. An isolated polypeptide having amolecular weight between 180 kD and 100 kD as determined by SDS-PAGE andcomprising the amino acid sequence AAGGILHLELLV.
 18. An isolatedpolypeptide according to claim 14, wherein said polypeptide furthercomprises the amino acid sequenceAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI directly following the amino acid sequence AAGGILHLELLV.19. An isolated polypeptide according to claim 18 having a molecularweight of about 170 kD.
 20. An isolated polypeptide according to claim18 having a molecular weight of about 160 kD.
 21. An isolatedpolypeptide according to claim 18 having a molecular weight of about 120kD.
 22. An isolated polypeptide according to claim 18 having a molecularweight of about 110 kD.
 23. A vWF cleaving complex comprising apolypeptide according to claim 18 and a divalent metal ion selected fromthe group consisting of Ca⁺⁺, Sr⁺⁺ and Ba⁺⁺.
 24. A vWF cleaving complexaccording to claim 23 wherein the divalent cation is Ca⁺⁺.
 25. A vWFcleaving complex comprising a complex according to claim 23, furthercontaining vWF.
 26. A composition comprising a polypeptide whichcomprises the sequence AAGGILHLELLV.
 27. Use of a composition accordingto claim 14 for the development of anti-peptide antibodies orderivatives thereof.
 28. A method of purifying von Willebrand factorcomprising contacting a solution containing von Willebrand factor with apolypeptide substrate comprising the amino acid sequence AAGGILHLELLVunder conditions sufficient to bind von Willebrand factor to thesubstrate.
 29. A composition according to claim 18, wherein the aminoacid sequence is encoded by the polynucleotide according to FIG.
 2. 30.An isolated polypeptide having vWF protease activity wherein saidpolypeptide comprises the amino acid sequenceAAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI.
 31. An isolated polypeptide according toclaim 30 wherein said polypeptide is encoded by a polynucleotidesequence according to FIG.
 2. 32. A host cell and progeny thereofcontaining a polynucleotide according to FIG.
 2. 33. A method for theproduction of a polypeptide exhibiting vWF protease activity comprisinggrowing, in a nutrient medium, a host cell comprising an expressionvector comprising, in the direction of transcription, a transcriptionalregulatory region and a translational initiation region functional in ahost cell, a cDNA sequence encoding for a polypeptide according to claim18, wherein said cDNA comprises the sequence according to FIG. 2 andtranscriptional and translational termination regions functional in saidhost cell, wherein the expression of said DNA is regulated by saidinitiation and termination regions, and isolating said polypeptide. 34.Use of a polypeptide according to claim 18 for the production of apreparation for the prophylaxis or therapy of thrombosis andthromboembolic diseases.
 35. Use according to claim 35, wherein thedisease can be selected from the group consisting of thromboticthrombocytic purpura (TTP), Henoch-Schönlein purpura, preeclampsia,neonatal thrombocytopenia or hemolyticuremic syndrome.